The present invention relates to a novel process to make indolo-carbazole carbazole glycosides in high purity which inhibit the growth of tumor cells and are therefore useful in the treatment of cancer in mammals, and the like.
In the field of cancer chemotherapy, a large number of compounds have already been put to practical use as antitumor agents. However, a need continues for the development of more efficacious compounds that work against a variety of tumors (see the Proceedings of the 47th General Meeting of the Japan Cancer Society, pp. 12-15 (1988)). This need has led to the development of indolocarbazole derivatives. (See U.S. Pat. Nos. 4,487,925; 4,552,842; 4,785,085; 5,591,842 and 5,922,860; Japanese Patent No. 20277/91; Journal of Antibiotics, Vol. 44, pp. 723-728 (1991); W091/18003; WO 98/07433; and EP0545195 A1). These compounds have been shown to act as topoisomerase inhibitors and therefore useful in the treatment of cancer (Cancer Chemother. Pharmacol. 34 (suppl): S41-S45 (1994)).
The success of these compounds in treating numerous cancers has necessitated the development of improved methods for their syntheses. (see Bioorg. and Med. Chem. Letters 2000, 10, 419; Tetrahedron 1997, 53, 5937; Tetrahedron 1997, 53, 585; and Synthesis 1976, 414). The previously known methods, however, suffer from numerous problems, including the use of undesirable solvents, mercury or silver salts, low yields and formation of unwanted side-products necessitating tedious or protracted purification steps.
For example, the previously known methods of preparation of the indolocarbazole glycoside III in high purity require purification procedures, such as combinations of carbon treatment, chromatography and/or recrystallization of the crude material, that are tedious, time-consuming and dangerous, especially when performed on a commercial scale due to the highly cytotoxic nature of the product. (see Bioorg and Med Chem Letters 1999, 3307; and Tetrahedron 1997, 585 (describing synthesis of structurally similar compound, requiring re-dissolution of crude product to obtain pure material)). 
An object of this invention therefore is to provide a novel route to these indolopyrrolocarbazole-derived antitumor substances while overcoming the problems inherent in the previously known syntheses, specifically a route producing the product in sufficient purity to allow for use xe2x80x9cas isxe2x80x9d in subsequent formulations.
The present invention relates to a novel process to make indolo-carbazole glycosides of Formula I in high purity which inhibit the growth of tumor cells and are therefore useful in the treatment of cancer in mammals, and the like. 
An embodiment of the present invention is illustrated by a process for the preparation of a compound of Formula I in high purity, 
which comprises the steps of:
(a) adjusting the pH of an acidic mixture consisting essentially of an alcohol, an acid, water and compound I, by adding a base to produce a solution with a pH in the range of about 1.5 to about 6.5;
(b) keeping the temperature of the solution from step (a) in the range of about 50xc2x0 C. to about 100xc2x0 C. ; and
(c) isolating the crystals of compound I.
In a second embodiment of the instant invention, the process comprises the steps of:
(a) adjusting the pH of an acidic mixture consisting essentially of an acid, alcohol, water and compound I, by adding a base to produce a solution with a pH in the range of about 1.5 to about 6.5;
(b) adjusting the solution from step (a) with an alcohol so that the solution is about 10% w/v to about 30% w/v water in an alcohol and the concentration of compound I is about 10 mL/g to 20 mL/g;
(c) adjusting the temperature of the solution from step (b) to a temperature in the range of about 50xc2x0 C. to about 100xc2x0 C. ;
(d) adding an alcohol to the solution from (c) such that the solution is diluted to about 3:2 (solution: alcohol);
(e) aging the solution from (d) at a temperature in the range of about 50xc2x0 C. to about 100xc2x0 C. until crystals of compound I are formed to produce a slurry; and
(f) isolating the crystals of compound I.
In a further embodiment of the second embodiment, the process further comprises deprotecting intermediate II: 
(wherein R is independently a hydrogen or a substituted or unsubstituted benzyl protecting group, with the proviso that at least one R is a substituted or unsubstituted benzyl protecting group), via hydrogenation in the presence of a catalyst to produce a reaction mixture, followed by filtering the reaction mixture to afford the mixture of step (a).
In an alternative embodiment, R is benzyl in the process described above.
In a third embodiment of the instant invention, the process for the preparation of compound m in high purity 
comprises the steps of:
(a) adjusting the pH of an acidic mixture consisting essentially of an acid, alcohol, water and compound III, by adding a base to produce a solution with a pH in the range of about 1.5 to about 6.5;
(b) adjusting the solution from step (a) with an alcohol so that the solution is about 10% w/v to about 30% w/v water in an alcohol and the concentration of compound III is about 10 mL/g to 20 mL/g;
(c) adjusting the temperature of the solution from step (b) to a temperature in the range of about 50xc2x0 C. to about 100xc2x0 C. ;
(d) seeding the solution;
(e) adding an alcohol to the solution such that the solution is diluted to about 3:2 (solution: alcohol);
(f) aging the solution from (e) at a temperature in the range of about 50xc2x0 C. to about 100xc2x0 C. until crystals of compound III are formed to produce a slurry; and
(g) isolating the crystals of compound III.
In a further embodiment of the third embodiment, the process further comprises deprotecting intermediate II: 
(wherein R is independently a hydrogen or a substituted or unsubstituted benzyl protecting group, with the proviso that at least one R is a substituted or unsubstituted benzyl protecting group), via hydrogenation in the presence of a catalyst to produce a reaction mixture, followed by filtering the reaction mixture to afford the mixture of step (a).
And yet another embodiment is the process described immediately above wherein the pH in step (a) is adjusted to a pH in the range of about 1.5 to about 3.5; the solution in (b) is adjusted so that the solution is about 15% w/v to about 25% w/v water in alcohol and the concentration of compound III is about 12 mL/g to about 18 mL/g; and the temperature in step (c) is adjusted to about 70xc2x0 C.
A further embodiment is the process above wherein the pH in step (a) is adjusted to a pH of about 2.5; the solution in (b) is adjusted so that the solution is about 20% w/v water in alcohol and the concentration of compound III is about 15 mL/g; and the temperature in step (c) is adjusted to about 70xc2x0 C.
Also encompassed by the present invention is the process described above further comprising the step of adjusting the slurry after step (f) such that the water content is lowered to a range of about 1% w/v to about 10% w/v before isolating the crystals of compound III in step (g).
A preferred embodiment is a process for the preparation of compound III in high purity, 
which comprises the steps of:
(a) adjusting the pH of an acidic mixture consisting essentially of an acid, alcohol, water and compound III, by adding a lower alkyl amine base to produce a solution with a pH of about 2.5;
(b) adjusting the solution from step (a) with isopropyl alcohol so that the solution is about 20% w/v water in isopropyl alcohol and the concentration of compound I is about 15 mL/g;
(c) adjusting the temperature of the solution from step (b) to a temperature of about 70xc2x0 C. ;
(d) seeding the solution;
(e) adding isopropyl alcohol to the solution such that the solution is diluted to about 3:2 (solution:isopropyl alcohol);
(f) aging the solution from (e) at a temperature of about 70xc2x0 C. until the crystals of compound I are formed to produce a slurry;
(g) adjusting the slurry so that the water content is about 3% w/v;
(h) aging the slurry at about 70xc2x0 C. before cooling down to about 22xc2x0 C. ; and
(i) isolating the crystals of compound III.
In a further embodiment of the preferred embodiment, the process further comprises deprotecting intermediate II: 
(wherein R is independently a hydrogen or a substituted or unsubstituted benzyl protecting group, with the proviso that at least one R is a substituted or unsubstituted benzyl protecting group), via hydrogenation in the presence of a catalyst to produce a reaction mixture, followed by filtering the reaction mixture to afford the mixture of step (a).
Also within the scope of the present invention is the process described immediately above wherein the lower alkyl amine in step (c) is triethylamine.
The present invention generates product which can be directly crystallized from the reaction medium without additional purification steps. In addition, the presently claimed process affords crystals with a more stable crystal geometry, 3-D trapezoid, than the previously known methods, 2-D needles, as suggested by solubility kinetics.
Compound I and Compound III may be synthesized following the procedure described in U.S. Pat. No. 5,591,842, issued Jan. 7, 1997, herein incorporated by reference. The powder of Compound III, which is obtained by the process described in WO 95130682 and U.S. Pat. No. 5,591,842, is crystallized by the method of the instant invention.
As used in the present application, xe2x80x9chigh purityxe2x80x9d refers to product which is obtained with 1% or less total impurities as measured by HPLC.
An xe2x80x9cacidic mixturexe2x80x9d refers to a mixture that has a pH that is less than about 7.0. Most preferably, the pH of the acidic mixture is less than about 2.5.
For the present application, types of acids that can be used include, but are not limited to, anhydrous or aqueous HF, HCl, HBr, HI, HNO3, HClO4, sulfuric, phosphoric, propionic, MsOH, TsOH, mono-phosphate salt, di-phosphate salt, mixed phosphate salt, carboxylic acids or ammonium halides. A mixed phosphate salt can be illustrated as M1M2HPO4, where M1 and M2 are independently selected from H, Na, K, NH4OH, sodium potassium, and the like. More preferably, the acids are selected from HCl.
The choice of catalyst in the hydrogenolysis reactions described herein will be readily apparent to the skilled artisan. Suitable catalysts include palladium on carbon, Pd(OH)2, Raney Nickel, tungsten catalysts, Rh/Al2O3, and the like. Palladium catalysts are preferred, such as palladium on carbon.
xe2x80x9cSeedingxe2x80x9d refers to the exposure of a solution to crystals (seed crystals) in order to catalyze the precipitation of crystals from the solution. Seeding can be done by addition of the seed crystals in a solid, dry form or the seed crystals may be added in the form of a slurry in a liquid. xe2x80x9cSeed crystalsxe2x80x9d may be crystals of the same compound being induced to precipitate or they may be of a different compound. In the present case, seeding with a slurry of the same compound is preferred.
xe2x80x9cAgingxe2x80x9d means to maintain the solution being aged at a constant temperature and volume for a certain period of time. The amount of time the reactions are aged in the present invention is not critical, unless specifically noted otherwise, and can be readily discerned by those skilled in the art.
xe2x80x9cFilteringxe2x80x9d means to pass the solution through some medium so that particulate matter is removed. The choice of medium is not critical and can be readily chosen by the average practitioner. Filtering may be accomplished by passing through celite, solka floc, sand, glass frit, diatomaceous earth, and the like.
xe2x80x9cAlcoholxe2x80x9dis intended to mean an organic molecule of 1 to 5 carbons, in a straight or branched chain, with at least on hydroxyl group as the prominent active group. Alcohol includes methanol, propanol, isopropanol, butanol, sec-butanol, etc. Isopropyl alcohol is the preferred alcohol.
The term xe2x80x9csubstituted benzyl protecting groupxe2x80x9d includes, but is not limited to, p-MeO-benzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl (wherein halo includes, chloro, bromo, and iodo), 2,6-dichlorobenzyl, diphenylmethyl, triphenylmethyl, and the like. Further suitable protecting groups may be found in Protective Groups in Organic Chemistry by Peter G. Wuts and Theodora W. Greene, John Wiley and Sons, 3rded. (1999).
The present invention comprises a step where the pH of a filtrate is adjusted to a particular range. The pH may be adjusted with any suitable base, such as triethylamine, dilsopropylethylamine, tributylamine, pyridine, 2,6-lutidine, 2,4,6-collidine, DBU, DBN, diisopropylamine, N,N-dimethylaniline, DABCO, N-alkylmorpholine, and the like. Lower alkyl amine bases are preferred. Triethylamine is the most preferred.
xe2x80x9cSlurryxe2x80x9d refers to a suspension of solid or crystals in a liquid. The solid may be partially, incompletely, or completely non-dissolved in the liquid.
Synopsis of Schemes
Scheme A illustrates a generalized approach to the preparation of biologically active indolocarbazole glycosides via a deprotection/crystallization protocol to afford product in high yield and high purity, obviating the need for further purification steps before formulation. 